DNA重组

1. If the trityl group is not removed from a newly-synthesized oligonucleotide it may interfere with polymerization reactions.2. Evaporate the oligonucleotide to dryness.3. Add 300 ml 80% Acetic acid. ...

1. Pour and polymerize a 20% polyacrylamide gel no Urea.2. Remove clamps. Rinse with water. Remove comb. Rinse top of gel well.3. Insert comb teeth down until teeth just touch the gel. Mount the plate ...

How to Calculate the Melting Temperature of Oligonucleotides The melting temperature Tm of an oligonucleotide is its most critically important value. The most reliable and accurate determination of me ...

UV Quantitation of DNA DNA absorbs ultraviolet light due to its highly conjugated nature. DNA may thus be easily quantitated in a UV spectrometer. Typically 1 OD260 (i.e. a solution having an absorban ...

Oligonucleotide VisualizationThis is a question asked quite a bit most recently (late September 1995) in the bionet.molbio.methds-reagnts newsgroup. Here are two methods and who submitted them. UV Sha ...

UV shadowing is a technique for visualizing nucleic acids separated on acrylamide/urea gels. The technique utilizes shortwave UV light (254 nm) and a fluor-coated TLC plate. We recommend UV shadowing ...

In situ immunodetection of 5-methylcytosine (5-mC) on squashed cells using anti-methylcytosine Cross-references are to Schwarzacher & Heslop-Harrison 2000. Practical in situ Hybridization. Bios Oxfor ...

Methylation analyis using restriction enzyme digestionContributed by Dr. A. GratchevThis is a classical method of methylation analysis based on the property of some restriction enzymes to be unable to ...

DNA-Methyltransferase AssayThis protocol was written by Jean-Pierre Issa based on Adams et al.* Kam-Wing Jair has made some useful shortcuts that work well if you are careful.Here is Kam-Wing Jair's ...

DNA methylation is an epigenetic event that affects cell function by altering gene expression and refers to the covalent addition of a methyl group catalyzed by DNA methyltransferase (DNMT) to the 5-c ...

Calcium Phosphate Transfection of Neurons in Primary CultureDeveloped by Hank Dudek and Zhengui Xia (11/96)Modified by Steve Finkbeiner (rev. 7/97)Departments of Neurology and Physiology UCSF/GIND415- ...

CaPO4 Transfection for Chromaffin Cells1. 2M CaCl2 - 11.1g CaCl2 (anhydrous) + 47.3 ml H2O (or 14.7g CaCl2-2H2O) Filter sterilize and store at 4'C 2. 150 mM Na2HPO4 -2.13g Na2HPO4 + 99.7 ml H2O (or 4. ...

Materials:LipofectamineIMDM containing 10% fetal bovine serum 1% glutamine 1% aaIMDM containing 1% glutamineIMDM containing 20% fetal bovine serum 1% glutamine 1% aaIn a six-well or 35 mm tissue cultu ...

1. Constitutive - those enzymes that are continuously synthesized in the cell (e.g. the enzymes of glycolysis or the lac repressor protein); 2 In ...

b-Galactosidase Activity Assay -- Marian Price-Carter 9/7/00Day 1: Start overnight cultures in assay medium.Negative control: cells lacking b-galactosidase such as LT2; positive control: cells with hi ...

CAT Assay Ref. Seed and Sheen Gene 67 271-277 (1988)Normal reaction is Chloramphenicol + Acetyl-CoA - Acetylchloramphenicol Assay uses butyryl-CoA (more hydrophobic) 14C-Cm+ Bu-CoA-Bu-14C-Cm Km (Cm) ...

InroductionSingle-cell electroporation (SCE) is a technique we have developed to deliver genes into individual cells within intact tissues although it may also be applicable to cells in disperse cultu ...

CAT ASSAY 1. Transfer cells to a 15 ml tube. 2. Add 5 ml TBS- to flasks shake & pour into tubes. 3. Spin down the cells @ 1k rpm for 5'. 4. Resuspend in 1 ml TBS- . 5. Transfer to 1.5 ml tubes. ...

CAT Enzyme Assay System With Reporter Lysis BufferChloramphenicol acetyltransferase (CAT) encoded by a bacterial drug-resistance gene inactivates chloramphenicol by acetylating the drug at one or both ...